Methods and compositions using complement fixing monoclonal antibody to human T cells

ABSTRACT

Hybrid cell line for production of monoclonal antibody to an antigen found on all normal human T cells and cutaneous T lymphoma cells. The hybrid is formed by fusing splenocytes from immunized CAF 1  mice with P3X63Ag8U1 myeloma cells. Diagnostic and therapeutic uses of the monoclonal antibody are also disclosed.

This application is a division of our copending application Ser. No.432,452, filed Oct. 4, 1982, now U.S. Pat. No. 4,515,893, which in turnis a division of application Ser. No. 33,669, filed Apr. 26, 1979, nowU.S. Pat. No. 4,361,549.

FIELD OF THE INVENTION

This invention relates generally to new hybrid cell lines and morespecifically to hybrid cell lines for production of complement-fixingmonoclonal antibody to an antigen found on all normal human T cells andcutaneous T lymphoma cells, to the antibody so produced, and totherapeutic and diagnostic methods and compositions employing thisantibody.

DESCRIPTION OF THE PRIOR ART

The fusion of mouse myeloma cells to spleen cells from immunized mice byKohler and Milstein in 1975 [Nature 256, 495-497 (1975)] demonstratedfor the first time that it was possible to obtain a continuous cell linemaking homogeneous (so-called "monoclonal") antibody. Since this seminalwork, much effort has been directed to the production of various hybridcells (called "hybridomas") and to the use of the antibody made by thesehybridomas for various scientific investigations. See, for example,Current Topics in Microbiology and Immunology, Volume 81--"LymphocyteHybridomas", F. Melchers, M. Potter, and N. Warner, Editors,Springer-Verlag, 1978, and references contained therein; C. J.Barnstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and W. F.Bodmer, Nature 276, 397-399 (November, 1978); Handbook of ExperimentalImmunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwell,1978, Chapter 25; and Chemical and Engineering News, Jan. 1, 1979, 15-17the subject hybridoma was deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockeville, Mar., 20852 on Apr. 26,1979, and assigned the ATCC accession number CRL 8001. These referencessimultaneously indicate the rewards and complications of attempting toproduce monoclonal antibody from hybridomas. While the general techniqueis well understood conceptually, there are many difficulties met andvariations required for each specific case. In fact, there is noassurance, prior to attempting to prepare a given hybridoma, that thedesired hybridoma will be obtained, that it will produce antibody ifobtained, or that the antibody so produced will have the desiredspecificity. The degree of success is influenced principally by the typeof antigen employed and the selection technique used for isolating thedesired hybridoma.

The attempted production of monoclonal antibody to human lymphocytecell-surface antigens has been reported only in a few instances. See,for example, Current Topics in Microbiology and Immunology, ibid, 66-69and 164-169. The antigens used in these reported experiments werecultured human lymphoblastoid leukemia and human chronic lymphocyticleukemia cell lines. Many hybridomas obtained appeared to produceantibody to various antigens on all human cells. None of the hybridomasproduced antibody against a predefined class of human lymphocytes.

It should be understood that there are two principal classes oflymphocytes involved in the immune system of humans and animals. Thefirst of these (the thymus-derived cell or T cell) is differentiated inthe thymus from haemopoietic stem cells. While within the thymus, thedifferentiating cells are termed "thymocytes." The mature T cells emergefrom the thymus and circulate between the tissues, lymphatics, and thebloodstream. These T cells form a large proportion of the pool ofrecirculating small lymphocytes. They have immunological specificity andare directly involved in cell-mediated immune responses (such as graftrejection) as effector cells. Although T cells do not secrete humoralantibodies, they are sometimes required for the secretion of theseantibodies by the second class of lymphocytes discussed below. Sometypes of T cells play a regulating function in other aspects of theimmune system. The mechanism of this process of cell cooperation is notyet completely understood.

The second class of lymphocytes (the bone marrow-derived cells or Bcells) are those which secrete antibody. They also develop fromhaemopoietic stem cells, but their differentiation is not determined bythe thymus. In birds, they are differentiated in an organ analogous tothe thymus, called the Bursa of Fabricius. In mammals, however, noequivalent organ has been discovered, and it is thought that these Bcells differentiate within the bone marrow.

It is now recognized that T cells are divided into at least severalsubtypes, termed "helper", "suppressor", and "killer" T cells, whichhave the function of (respectively) promoting a reaction, suppressing areaction, or killing (lysing) foreign cells. These subclasses are wellunderstood for murine systems, but they have only recently beendescribed for human systems. See, for example, R. L. Evans, et al.,Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L.Chess and S. F. Schlossman--"Functional Analysis of Distinct HumanT-Cell Subsets Bearing Unique Differentiation Antigens", in"Contemporary Topics in Immunobiology", O. Stutman, Editor, PlenumPress, 1977, Volume 7, 363-379.

The ability to identify or suppress classes or subclasses of T cells isimportant for diagnosis or treatment of various immunoregulatorydisorders or conditions.

For example, certain leukemias and lymphomas have differing prognosisdepending on whether they are of B cell or T cell origin. Thus,evaluation of the disease prognosis depends upon distinguishing betweenthese two classes of lymphocytes. See, for example, A. C. Aisenberg andJ. C. Long, The American Journal of Medicine, 58:300 (March, 1975); D.Belpomme, et al., in "Immunological Diagnosis of Leukemias andLymphomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977,33-45; and D. Belpomme, et al., British Journal of Haematology, 1978,38, 85. Certain disease states (e.g., juvenile rheumatoid arthritis andcertain leukemias) are associated with an imbalance of T cellsubclasses. It has been suggested that autoimmune diseases generally areassociated with an excess of "helper" T cells or a deficiency of certain"suppressor" T cells, while malignancies generally are associated withan excess of "suppressor" T cells. In certain leukemias, excess T cellsare produced in an arrested stage of development. Diagnosis may thusdepend on the ability to detect this imbalance or excess. See, forexample, J. Kersey, et al., "Surface Markers Define Human LymphoidMalignancies with Differing Prognoses" in Haematology and BloodTransfusion, Volume 20, Springer-Verlag, 1977, 17-24, and referencescontained therein.

On the therapeutic side, there is some suggestion, as yet not definitelyproven, that administration of antibodies against the subtype of T cellin excess may have therapeutic benefit in autoimmune disease ormalignancies. Antisera against the entire class of human T cells(so-called antihuman thymocyte globulin or ATG) has been reported usefultherapeutically in patients receiving organ transplants. Since thecell-mediated immune response (the mechanism whereby transplants arerejected) depends upon T cells, administration of antibody to T cellsprevents or retards this rejection process. See, for example, Cosimi, etal., "Randomized Clinical Trial of ATG in Cadaver Renal AllgraftRecipients: Importance of T Cell Monitoring", Surgery 40:155-163 (1976)and references contained therein.

The identification and suppression of human T cell classes andsubclasses has previously been accomplished by the use of spontaneousautoantibodies or selective antisera for human T cells obtained byimmunizing animals with human T cells, bleeding the animals to obtainserum, and adsorbing the antiserum with (for example) autologous but notallogeneic B cells to remove antibodies with unwanted reactivities. Thepreparation of these antisera is extremely difficult, particularly inthe adsorption and purification steps. Even the adsorbed and purifiedantisera contain many impurities in addition to the desired antibody,for several reasons. First, the serum contains millions of antibodymolecules even before the T cell immunization. Second, the immunizationcauses production of antibodies against a variety of antigens found onall human T cells injected. There is no selective production of antibodyagainst a single antigen. Third, the titer of specific antibody obtainedby such methods is usually quite low, (e.g., inactive at dilutionsgreater than 1:100) and the ratio of specific to non-specific antibodyis less than 1/10⁶.

See, for example, the Chess and Schlossman article referred to above (atpages 365 and following) and the Chemical and Engineering News articlereferred to above, where the deficiencies of prior art antisera and theadvantages of monoclonal antibody are described.

SUMMARY OF THE INVENTION

There has now been discovered a novel hybridoma (designated OKT3) whichis capable of producing novel complementfixing monoclonal antibodyagainst an antigen found on essentially all normal human peripheral Tcells and cutaneous T lymphoma cells. The antibody so produced ismono-specific for a single determinant on normal human T cells andcutaneous T lymphoma cells and contains essentially no other anti-humanimmuneglobulin, in contrast to prior art antisera (which are inherentlycontaminated with antibody reactive to numerous human antigens) and toprior art monoclonal antibodies (which are not monospecific for a humanT cell antigen). Moreoever, this hybridoma can be cultured to produceantibody without the necessity of immunizing and killing animals,followed by the tedious adsorption and purification steps necessary toobtain even the impure antisera of the prior art.

It is accordingly one object of this invention to provide hybridomaswhich produce antibodies against an antigen found on essentially allnormal human T cells and cutaneous T lymphoma cells.

It is a further aspect of the present invention to provide methods forpreparing these hybridomas.

A further object of the invention is to provide essentially homogeneousantibody against an antigen found on essentially all normal human Tcells and cutaneous T lymphoma cells.

A still further object is to provide methods for treatment or diagnosisof disease employing these antibodies.

Other objects and advantages of the invention will become apparent fromthe examination of the present disclosure.

In satisfaction of the foregoing objects and advantages, there isprovided by this invention a novel hybridoma producing novel antibody toan antigen found on essentially all normal human T cells and cutaneous Tlymphoma cells, the antibody itself, and diagnostic and therapeuticmethods employing the antibody. The hybridoma was prepared generallyfollowing the method of Milstein and Kohler. Following immunization ofmice with normal E rosette positive human T cells, the spleen cells ofthe immunized mice were fused with cells from a mouse myeloma line andthe resultant hybridomas screened for those with supernatants containingantibody which gave selective binding to normal E rosette positive humanT cells. The desired hybridomas were subsequently cloned andcharacterized. As a result, a hybridoma was obtained which producesantibody (designated OKT3) against an antigen on essentially all normalhuman T cells. Not only does this antibody react with essentially allnormal human peripheral T cells, but it also does not react with othernormal peripheral blood lymphoid cells. In addition, the cell surfaceantigen recognized by this antibody is detected on only maturethymocytes and is completely lacking on greater than 90% of normal humanthymocytes.

In view of the difficulties indicated in the prior art and the lack ofsuccess reported using malignant cell lines as the antigen, it wassurprising that the present method provided the desired hybridoma. Itshould be emphasized that the unpredictable nature of hybrid cellpreparation does not allow one to extrapolate from one antigen or cellsystem to another. In fact, the present applicants have discovered thatusing a T cell malignant cell line as the antigen caused formation ofhybridomas which did not produce the desired antibody. Attempts to usepurified antigens separated from the cell surfaces were alsounsuccessful.

Both the subject hybridoma and the antibody produced thereby areidentified herein by the designation "OKT3", the particular materialreferred to being apparent from the context.

The preparation and characterization of the hybridoma and the resultantantibody will be better understood by reference to the followingdescription and Examples.

DETAILED DESCRIPTION OF THE INVENTION

The method of preparing the hybridoma generally comprises the followingsteps:

A. Immunizing mice with E rosette positive purified normal humanperipheral T cells. While it has been found that female CAF₁ mice (afirst generation hybrid between Balb/cJ and A/J mice) are preferred, itis contemplated that other mouse strains could be used. The immunizationschedule and T cell concentration should be such as to produce usefulquantities of suitably primed splenocytes. Three immunizations atfourteen day intervals with 2×10⁷ cells/mouse/injection in 0.2 mlphosphate buffered saline has been found to be effective.

B. Removing the spleens from the immunized mice and making a spleensuspension in an appropriate medium. About one ml of medium per spleenis sufficient. These experimental techniques are well-known.

C. Fusing the suspended spleen cells with mouse myeloma cells from asuitable cell line by the use of a suitable fusion promoter. Thepreferred ratio is about 5 spleen cells per myeloma cell. A total volumeof about 0.5-1.0 ml of fusion medium is appropriate for about 10⁸splenocytes. Many mouse myeloma cell lines are known and available,generally from members of the academic community or various depositbanks, such as the Salk Institute Cell Distribution Center, La Jolla,CA. The cell line used should preferably be of the so-called "drugresistant" type, so that unfused myeloma cells will not survive in aselective medium, while hybrids will survive. The most common class is8-azaguanine resistant cell lines, which lack the enzyme hypoxanthineguanine phophoribosyl transferase and hence will not be supported by HAT(hypoxanthine, aminopterin, and thymidine) medium. It is also generallypreferred that the myeloma cell line used be of the so-called"non-secreting" type, in that it does not itself produce any antibody,although secreting types may be used. In certain cases, however,secreting myeloma lines may be preferred. While the preferred fusionpromoter is polyethylene glycol having an average molecular weight fromabout 1000 to about 4000 (commercially available as PEG 1000, etc.),other fusion promoters known in the art may be employed.

D. Diluting and culturing in separate containers, the mixture of unfusedspleen cells, unfused myeloma cells, and fused cells in a selectivemedium which will not support the unfused myeloma cells for a timesufficient to allow death of the unfused cells (about one week). Thedilution may be a type of limiting one, in which the volume of diluentis statistically calculated to isolate a certain number of cells (e.g.,1-4) in each separate container (e.g., each well of a microtiter plate).The medium is one (e.g., HAT medium) which will not support thedrug-resistant (e.g., 8-azaguanine resistant) unfused myeloma cell line.Hence, these myeloma cells perish. Since the unfused spleen cells arenon-malignant, they have only a finite number of generations. Thus,after a certain period of time (about one week) these unfused spleencells fail to reproduce. The fused cells, on the other hand, continue toreproduce because they possess the malignant quality of the myelomaparent and the ability to survive in the selective medium of the spleencell parent.

E. Evaluating the supernatant in each container (well) containing ahybridoma for the presence of antibody to E rosette positive purifiedhuman T cells.

F. Selecting (e.g., by limiting dilution) and cloning hybridomasproducing the desired antibody.

Once the desired hybridoma has been selected and cloned, the resultantantibody may be produced in one of two ways. The purest monoclonalantibody is produced by in vitro culturing of the desired hybridoma in asuitable medium for a suitable length of time, followed by recovery ofthe desired antibody from the supernatant. The suitable medium andsuitable length of culturing time are known or are readily determined.This in vitro technique produces essentially monospecific monoclonalantibody, essentially free from other specific antihuman immuneglobulin. There is a small amount of other immune globulin present sincethe medium contains xenogeneic serum (e.g., fetal calf serum). However,this in vitro method may not produce a sufficient quantity orconcentration of antibody for some purposes, since the concentration ofmonoclonal antibody is only about 50 μg/ml.

To produce a much greater concentration of slightly less pure monoclonalantibody, the desired hybridoma may be injected into mice, preferablysyngenic or semisyngenic mice. The hybridoma will cause formation ofantibody-producing tumors after a suitable incubation time, which willresult in a high concentration of the desired antibody (about 5-20mg/ml) in the bloodstream and peritoneal exudate (ascites) of the hostmouse. Although these host mice also have normal antibodies in theirblood and ascites, the concentration of these normal antibodies is onlyabout 5% of the monoclonal antibody concentration. Moreover, since thesenormal antibodies are not antihuman in their specificity, the monoclonalantibody obtained from the harvested ascites or from the serum isessentially free of any contaminating antihuman immune globulin. Thismonoclonal antibody is high titer (active at dilutions of 1:100,000 orhigher) and high ratio of specific to non-specific immune globulin(about 1/20). Immune globulin produced incorporating the κ light myelomachains are non-specific, "nonsense" peptides which merely dilute themonoclonal antibody without detracting from its specificity.

EXAMPLE I Production of Monoclonal Antibodies

A. Immunization and Somatic Cell Hybridization

Female CAF₁ mice (Jackson Laboratories; 6-8 weeks old) were immunizedintraperitoneally with 2×10⁷ E rosette purified T cells in 0.2 ml ofphosphate buffered saline at 14-day intervals. Four days after the thirdimmunization, spleens were removed from the mice, and a single cellsuspension was made by pressing the tissue through a stainless steelmesh.

Cell fusion was carried out according to the procedure developed byKohler and Milstein. 1×10⁸ splenocytes were fused in 0.5 ml of a fusionmedium comprising 35% polyethylene glycol (PEG 1000) and 5%dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with2×10⁷ P3X63Ag8U1 myeloma cells supplied by Dr. M. Scharff, AlbertEinstein College of Medicine, Bronx, NY. These myeloma cells secreteIgG₁ κ light chains.

B. Selection and Growth of Hybridoma

After cell fusion, cells were cultured in HAT medium (hypoxanthine,aminopterin, and thymidine) at 37° C. with 5% CO₂ in a humid atmosphere.Several weeks later, 40 to 100 μl of supernatant from culturescontaining hybridomas were added to a pellet of 10⁶ peripherallymphocytes separated into E rosette positive [E ) and E rosettenegative (E⁻) populations, which were prepared from blood of healthyhuman donors as described by Mendes (J. Immunol. 111:860, 1973).Detection of mouse hybridoma antibodies binding to these cells wasdetermined by radioimmunoassay and/or indirect immunofluorescence. Inthe first method, the cells were initially reacted with 100 μl ofaffinity-purified ¹²⁵ I goat-anti-mouse IgG (10⁶ cpm/μg; 500 μg/μl ).(Details of iodination of goat-anti-mouse IoG were described by Kung, etal., J. Biol. Chem. 251(8):2399, 1976). Alternatively, cells incubatedwith culture supernatants were stained with a fluorescinatedgoat-anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/p=2.5) and the fluorescent antibody-coated cells were subsequentlyanalyzed on the Cytofluorograf FC200/4800A (Ortho Instruments, Westwood,MA) as described in Example III. Hybridoma cultures containingantibodies reacting specifically with E⁺ lymphocytes (T cells) wereselected and cloned. Subsequently, the clones were transferredintraperitoneally by injecting 1×10⁷ cells of a given clone (0.2 mlvolume) into CAF₁ mice primed with 2,6,10,14-tetramethylpentadecane,sold by Aldrich Chemical Company under the name Pristine. The malignantascites from these mice were then used to characterize lymphocytes asdescribed below in Example II. The subject hybrid antibody OKT3 wasdemonstrated by standard techniques to be of IgG₂ subclass and to fixcomplement.

EXAMPLE II Characterization of OKT3 Reactivity

A. Isolation of Lymphocyte Populations

Human peripheral blood mononuclear cells were isolated from healthyvolunteer donors (ages 15-40) by FicollHypaque density gradientcentrifugation (Pharmacia Fine Chemicals, Piscataway, NJ) following thetechnique of Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77,1968. Unfractionated mononuclear cells were separated into surface Ig⁺(B) and Ig⁻ (T plus Null) populations by Sephadex G-200 anti-F(ab')₂column chromatography as previously described by Chess, et al., J.Immunol. 113:1113 (1974). T cells were recovered by E rosetting the Ig⁻population with 5% sheep erythrocytes (Microbiological Associates,Bethesda, MD). The rosetted mixture was layered over Ficoll-Hypaque andthe recovered E⁺ pellet treated with 0.155M NH₄ Cl (10⁸ ml per cells).The T cell population so obtained was <2% EAC rosette positive and >95%E rosette positive as determined by standard methods. In addition, thenon-rosetting Ig⁻ (Null cell) population was harvested from the Ficollinterface. This latter population was <5% E⁺ and ≦2% sIg⁺. The surfaceIg⁺ (B) population was obtained from the Sephadex G-200 column followingelution with normal human gamma globulin as previously described. Thispopulation was >95% surface Ig⁺ and <5% E⁺.

Normal human macrophages were obtained from the mononuclear populationby adherence to polystyrene. Thus, mononuclear cells were resuspended infinal culture media (RPMI 1640, 2.5 mM HEPES[4-(2-hydroxyethyl)-1-piperazinepropane sulfonic acid]buffer, 0.5%sodium bicarbonate, 200 mM L-glutamine, and 1% penicillinstreptomycin,supplemented with 20% heat-inactivated human AB serum) at aconcentration of 2×10⁶ cells and incubated in plastic petri dishes(100×20 mm) (Falcon Tissue Culture Dish; Falcon, Oxnard, CA) at 37° C.overnight. After extensive washing to remove non-adherent cells, theadherent population was detached by brisk washing with cold serum-freemedium containing 2.5 mM EDTA and occasional scraping with the rubbertip of a disposable syringe plunger. Greater than 85% of the cellpopulation was capable of ingesting latex particles and had morphologiccharacteristics of monocytes by Wright-Giemsa staining.

B. Normal Thymus

Normal human thymus gland was obtained from patients aged two months to14 years undergoing corrective cardiac surgery. Freshly obtainedportions of the thymus gland were immediately placed in 5% fetal calfserum in medium 199 (Gibco), finely minced with forceps and scissors,and subsequently made into single cell suspensions by being pressedthrough wire mesh. The cells were next layered over Ficoll-Hypaque andspun and washed as previously described in section A above. Thethymocytes so obtained were >95% viable and ≧90% E rosette positive.

C. Cell Lines

Epstein-Barr Virus (EBV) transformed B cell lines from four normalindividuals (Iaz 007, Laz 156, Laz 256, and SB) and described. T celllines CEM, HJD-1, Laz 191, and HMl established from leukemic patientswere provided by Dr. H. Lazarus, Sidney Farber Cancer Institute, Boston,MA.

D. .T Acute Lymphoblastic Leukemia (T-ALL) Cells and T Chronic LymphaticLeukemia (T-CLL) Cells

Leukemia cells were obtained from 12 patients with T-ALL. Theseindividuals' cells had previously been determined to be of T celllineage by their spontaneous rosette formation with sheep erythrocytes(>20% E⁺) reactivity with T cell specific hetero-antisera, antiHTL(anti-B.K.) and A99, as previously described by Schlossman, et al.,Proc. Nat. Acad. Sci. 73:1288 (1976). Tumor cells from three individualswere reactive (TH₂ ⁺) with rabbit and/or equine anti-TH₂ while cellsfrom the remaining nine were non-reactive (TH₂ ) Leukemic cells from twopatients with TH₂ ⁻ T-CLL were also utilized. Both acute and chronic Tcell leukemia cells were cryopreserved in -196° C. vapor phase liquidnitrogen in 10% dimethylsulfoxide and 20% AB human serum until the timeof surface characterization. The tumor populations analyzed were >90%blasts by Wright-Giemsa morphology in all instances.

EXAMPLE III Cytofluorographic Analysis

Cytofluorographic analysis of all cell populations was performed byindirect immunofluorescence with fluorescein-conjugated goat-anti-mouseIgG (G/M FITC) (Meloy Laboratories) on a Cytofluorograf FC200/4800A(Ortho Instruments). In brief, 1-2×10⁶ cells were treated with 0.15 mlOKT3 at a 1:1000 dilution, incubated at 4° C. for 30 minutes, and washedtwice. The cells were then reacted with 0.15 ml of a 1:40 dilution G/MFITC at 4° C. for 30 minutes, centrifuged, and washed three times. Thesecells were then analyzed on the Cytofluorograf and the intensity offluorescence per cell recorded on a pulse height analyzer. A similarpattern of reactivity was observed at a dilution of 1:100,000, butfurther dilution caused loss of reactivity. Background staining wasobtained by substituting a 0.15 ml aliquot of 1:1000 ascites from aBalb/cJ mouse intraperitoneally immunized with a non-producing hybridclone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fluorescence pattern obtained on the Cytofluorografafter reacting normal human peripheral T cells with OKT3 at a 1:1000dilution and G/M FITC. For comparison, results with monoclonalantibodies OKTl and OKT4 are shown under equivalent conditions in FIGS.1-5.

FIG. 2 shows the fluorescence pattern obtained on the Cytofluorografafter reacting human thymocytes with OKT3 and G/M FITC.

FIG. 3 shows the fluorescence pattern obtained on the Cytofluorografafter reacting leukemic cells from B cell chronic lymphoblastic leukemiapatients with OKT3 and G/M FITC.

FIG. 4 shows the fluorescence pattern obtained on the Cytofluorografafter reacting the human T cell line HJD-1 with OKT3 and G/M FITC.

FIG. 5 shows the fluorescence pattern obtained on the Cytofluorografafter reacting the human T cell line CEM with OKT3 and G/M FITC.

The data in FIGS. 1-5 plus additional data for OKT3 (as well as OKTl andOKT4) are summarized in Table I.

The production of the hybridoma and the production and characterizationof the resulting monoclonal antibody were conducted as described in theabove Examples. Although large quantities of the subject antibody wereprepared by injecting the subject hybridoma intraperitoneally into miceand harvesting the malignant ascites, it is clearly contemplated thatthe hybridoma could be cultured in vitro by techniques well-known in theart and the antibody removed from the supernatant.

As shown in FIG. 1, the entire human peripheral blood T cell populationof a given normal individual is reactive with OKT3, whereas the entire Bcell, null cell, and macrophage populations isolated from the sameindividual are unreactive with OKT3. Similar results were obtained onpopulations of lymphocytes from fifteen other normal individuals. Themonoclonal antibody is thus characterized in that it is reactive with anantigen contained on the surface of essentially all normal humanperipheral T cells, while being unreactive with any antigens on thesurface of the other three cell types discussed above. This differentialreactivity is one test by which the subject antibody OKT3 may bedetected and distinguished from other antibodies.

As shown in FIG. 2, the vast majority of normal human thymocytes from asix-month old infant are completely unreactive with OKT3, while about 5to 10 percent of the thymocytes are reactive. The implication of thisfinding is that, during the differentiation process by which stem cellsare converted into mature T cells, the thymocytes acquire at some stagethe same surface antigen found on T cells, which is reactive with OKT3.It is believed that these thymocytes are in the later stages ofdifferentiation just prior to emergence from the thymus into thebloodstream. Similar results (5-10% reactivity) were obtained using sixadditional thymus specimens from normal individuals two months to 19years of age. The pattern of reactivity in FIG. 2 provides a secondmethod of detecting the subject antibody OKT3 and distinguishing it fromother antibodies.

The subject antibody is also useful for determining the proportion ofcirculating lymphocytes that are T cells. As shown in Table I, ≧95% ofall T cells react with OKT3 antibody. The present invention thusincludes a method for determining in an individual the proportion ofcirculating lymphocytes that are T cells which comprises mixing OKT3antibody with a lymphocyte composition from the individual anddetermining the preparation of the lymphocytes which are OKT3⁺, and thusT cells.

A further characterization of the subject antibody OKT3 is shown by thereactivity to various human T cell lines illustrated in FIGS. 4 and 5.As can be seen, the reactivity of the subject antigen to human T celllines was heterogeneous, being weak for the line HJD-1, and nonexistentfor the lines CEM, Laz 191, and HMl. This differential reactivity ofOKT3 to various readilyavailable human T cell lines provides yet anothermethod of characterizing and describing the subject antibody. The lackof reaction of OKT3 with the human B cell lines Laz 007, Laz 156, Laz256, and SB is shown in Table I. This further supports the lack ofreactivity of OKT3 with B cells obtained from the peripheral blood of anormal human population and provides yet another method forcharacterizing and distinguishing the subject antibody OKT3.

The specific reaction of OKT3 antibody with an antigen on cutaneous Tcell lymphomas is illustrated by Table II, where the distinction fromOKTl and OKT4 is shown. The present antibody thus provides a reagent forconfirming a diagnosis of cutaneous T cell lymphoma in a patientsuspected of having said disease. Treatment of cutaneous T cell lymphomaby administration of a therapeutically effective amount of OKT3 antibodyis also contemplated as part of the present invention.

According to the present invention there are provided a hybridomacapable of producing antibody against an antigen found on essentiallyall normal human T cells and cutaneous T lymphoma cells, a method forproducing this hybridoma, monoclonal antibody against an antigen foundon essentially all human T cells, methods for producing the antibody,and methods for treatment or diagnosis of disease employing thisantibody.

Although only a single hybridoma producing a single monoclonal antibodyagainst human T cell antigen is described, it is contemplated that thepresent invention encompasses all monoclonal antibodies exhibiting thecharacteristics described herein. It was determined that the subjectantibody OKT3 belongs to the subclass IgG₂, which is one of foursubclasses of murine IgG. These subclasses of immune globulin G differfrom one another in the so-called "fixed" regions, although an antibodyto a specific antigen will have a so-called "variable" region which isfunctionally identical regardless of which subclass of immune globulin Git belongs to. That is, a monclonal antibody exhibiting thecharacteristic described herein may be of subclass IgG₁, IgG₂ a, IgG₂ b,or IgG₃, or of classes IgM, IgA, or other known Ig classes. Thedifferences among these classes or subclasses will not affect theselectivity of the reaction pattern of the antibody, but may affect thefurther reaction of the antibody with other materials, such as (forexample) complement or anti-mouse antibodies. Although the subjectantibody is specifically IgG₂, it is contemplated that antibodies havingthe patterns of reactivity illustrated herein are included within thesubject invention regardless of the immune globulin class or subclass towhich they belong.

Further included within the subject invention are methods for preparingthe monoclonal antibodies described above employing the hybridomatechnique illustrated herein. Although only one example of a hybridomais given herein, it is contemplated that one skilled in the art couldfollow the immunization, fusion, and selection methods provided hereinand obtain other hybridomas capable of producing antibodies having thereactivity characteristics described herein. Since the individualhybridoma produced from a known mouse myeloma cell line and spleen cellsfrom a known species of mouse cannot be further identified except byreference to the antibody produced by the hybridoma, it is contemplatedthat all hybridomas producing antibody having the reactivitycharacteristics described above are included within the subjectinvention, as are methods for making this antibody employing thehybridoma.

Further aspects of the invention are methods of treatment or diagnosisof disease employing the monoclonal antibody OKT3 or any othermonoclonal antibody exhibiting the pattern of reactivity providedherein. As discussed above, the subject antibody allows treatment ofpatients having certain T cell chronic lymphoblastic leukemias byadministration of a therapeutically-effective amount thereof.Administration of a therapeutically-effective amount of OKT3 antibody toan individual subject undergoing organ transplant will reduce oreliminate the rejection of this transplant. The subject antibody alsoallows detection of cutaneous T cell lymphoma in an individual by mixinga lymphoma T cell composition from said individual with adiagnostically-effective amount of OKT3 antibody. The presence of areaction confirms the identity of the disease. The cutaneous T celllymphoma may be treated by administering to an individual in need ofsuch treatment a therapeutically-effective amount of OKT3 antibody. Thisantibody will react with and reduce the amount of T lymphoma cells, thusameliorating the disease. In view of these diagnostic and therapeuticmethods, the present invention additionally includes diagnostic andtherapeutic compositions comprising (respectively) adiagnostically-effective or therapeutically-effective amount of OKT3antibody in a diagnostically or pharmaceutically acceptable carrier.

                  TABLE I                                                         ______________________________________                                        MONOCLONAL ANTIBODY REACTIVITY                                                AND PROPERTIES                                                                               Monoclonal Antibodies                                                         OKT1    OKT3      OKT4                                         ______________________________________                                        % Reactivity With:                                                            Peripheral T-cells (10 samples)                                                                >95%      >95%      55%                                      Peripheral B-cells (10 samples)                                                                <2%       <2%       <2%                                      Peripheral Null cells                                                                          <2%       <2%       <2%                                      (10 samples)                                                                  Thymocytes* (8 samples)                                                                        5-10%     5-10%     80%                                      Reactivity With:                                                              T-chronic lymphatic Leukemia                                                                   +         +(1);-(2) -                                        (3 cases)                                                                     T-acute lymphatic Leukemia                                                                     -         -         -                                        (8 cases)                                                                     Null acute lymphatic Leukemia                                                                  -         -         -                                        (15 cases)                                                                    B-chronic lymphatic Leukemia                                                                   +(4);-(2) -         -                                        (6 cases)                                                                     B-cell lines.sup.+  (4)                                                                        -         -         -                                        T-cell lines.sup.+  HJD-1                                                                      +         (±)    -                                        CEM              +         -         +                                        Laz 191          +         -         -                                        HM1              +         -         -                                        IgG Subclass     IgG.sub.1 IgG.sub.2 IgG.sub.2                                Complement fixation                                                                            -         +         +                                        ______________________________________                                         *From patients aged 2 months to 18 years                                      +Obtained from Dr. H. Lazarus, Sidney Farber Cancer Center. B cell lines      Laz 256, 156, 007 and SB obtained by EpsteinBarr virus transformation of      human peripheral B cells and HJD1, CEM, Laz 191, and HM1 established from     leukemic patients.                                                       

                  TABLE II                                                        ______________________________________                                                  Cutaneous T-                                                                              MONOCLONAL                                              Patient's Cell Lymphoma                                                                             ANTIBODY ASSAYS                                         Name      DIAGNOSIS   OKT1      OKT3  OKT4                                    ______________________________________                                        E. McBride                                                                              Sezary Blast                                                                              +         +     -                                                 Crisis; PBL                                                         C. O. Okley                                                                             Mycosis     -         +     +                                                 Fungoides;                                                                    Node                                                                Odom      Mycosis     +         +     -                                                 Fungoides;                                                                    Node                                                                Montalbono                                                                              ?           +         +     +                                                 Node                                                                ______________________________________                                         Source of cells: PBL = peripheral blood lymphocytes                           Node = lymph node                                                        

What is claimed is:
 1. A therapeutic composition of matter comprising,in admixture with a pharmaceutically-acceptable carrier, atherapeutically effective amount of a mouse monoclonal antibody, saidamount being effective to reduce or eliminate the rejection of atransplant by an organ transplant recipient, said monoclonal antibodyfixing complement and reacting with essentially all normal humanperipheral T cells, but not with normal human peripheral B cells, nullcells, or macrophages.
 2. A therapeutic composition of mattercomprising, in admixture with a pharmaceutically-acceptable carrier, atherapeutically effective amount of a mouse monoclonal antibody, saidamount being effective to reduce or eliminate the rejection of atransplant by an organ transplant recipient, said monoclonal antibodyfixing complement and reacting with essentially all normal humanperipheral T cells.
 3. A method of treatment of an organ transplantrecipient to reduce or eliminate rejection of said transplanted organwhich comprises administration to said recipient of an amount of mousemonoclonal antibody effective to cause said reduction or elimination,said antibody fixing complement and reacting with essentially all normalhuman peripheral T cells but not with normal human peripheral B cells,null cells, or macrophages.
 4. A therapeutic composition of mattercomprising, in admixture with a pharmaceutically acceptable carrier, atherapeutically-effective amount of monoclonal antibody produced from ahydriboma having the identifying characteristics of ATCC CRL 8001 saidamount being effective to reduce or eliminate the rejection of atransplant by an organ transplant recipient.
 5. A therapeuticcomposition of matter comprising, in admixture with a pharmaceuticallyacceptable carrier, a therapeutically-effective amount of monoclonalantibody, said amount being effective to reduce or eliminate therejection of a transplant by an organ transplant recipient, whichantibody:(a) reacts with essentially all normal human peripheral T cellsand cutaneous T lymphoma cells, but not with normal human peripheral Bcells, null cells or macrophages; (b) reacts with from about 5% to about10% of normal human thymocytes; (c) reacts with leukemic cells fromhumans with T cell chronic lymphoblastic leukemia but does not reactwith leukemic cells from humans with T cell acute lymphoblasticleukemia, null cell acute lymphoblastic leukemia, or B cell chroniclumphatic leukemia; (d) reacts weakly with the human T cell line HJD-1but does not react with CEM, Laz 191, or HMl; (e) does not react withthe Epstein-Barr virus-transformed human B cell lines Laz 007, Laz 156,Laz 256, or SB; and (f) fixed complement.
 6. A method of treatment of anorgan transplant recipient to reduce or eliminate rejection of saidtransplanted organ which comprises administration to said recipient ofan amount of mouse monoclonal antibody effective to cause said reductionor elimination, said antibody fixing complement and reacting withessentially all normal human peripheral T cells.
 7. A method oftreatment of an organ transplant recipient to reduce or eliminateallograft rejection of said transplanted organ which comprisesadministration of an amount of monoclonal antibody effective to causesaid reduction or elimination, which antibody:(a) reacts withessentially all normal human peripheral T cells and cutaneous T lymphomacells, but not with normal human peripheral B cells, null cells ormacrophages; (b) reacts with from about 5% to about 10% of normal humanthymocytes; (c) reacts with leukemic cells from humans with T cellchronic lymphoblastic leukemia but does not react with leukemic cellsfrom humans with T cell acute lymphoblastic leukemia, null cell acutelymphoblastic leukemia, or B cell chronic lymphatic leukemia; (d) reactsweakly with the human T cell line HJD-1 but does not react with CEM, Laz191, or HM1; (e) does not react with the Epstein-Barr virus-transformedhuman B cell lines Laz 007, Laz 156, Laz 256, or SB; and (f) fixescomplement.
 8. The method of claim 7 wherein the antibody is producedfrom a hybridoma having the identifying characteristics of ATCC CRL8001.
 9. A diagnostic composition for determining in an individual theproportion of circulating lymphocytes that are T cells which comprises adiagnostically-effective amount of a mouse monoclonal antibody thatfixed complement and reacts with essentially all normal human peripheralT cells, but not with normal human peripheral B cells, null cells, ormacrophages, said antibody being admixed with a diagnosticallyacceptable carrier.
 10. A diagnostic composition for determining in anindividual the proportion of circulating lymphocytes that are T cellswhich comprises a diagnostically-effective amount of a mouse monoclonalantibody that fixed complement and reacts with essentially all normalhuman peripheral T cells, said antibody being admixed with adiagnostically acceptable carrier.
 11. A diagnostic composition fordetermining in an individual the proportion of circulating lymphocytesthat are T cells which comprises a diagnostically-effective amount of amouse monoclonal antibody that reacts with essentially all normal humanperipheral T cells, but not with normal human peripheral B cells, nullcells, or macrophages, said antibody being admixed with a diagnosticallyacceptable carrier.
 12. A diagnostic composition for determining in anindividual the proportion of circulating lymphocytes that are T cellswhich comprises a diagnostically-effective amount of a mouse monoclonalantibody that reacts with essentially all normal human peripheral Tcells, said antibody being admixed with a diagnostically acceptablecarrier.
 13. A diagnostic composition for determining in an individualthe proportion of circulating lymphocytes that are T cells whichcomprises a diagnostically-effective amount of a mouse monoclonalantibody having the identifying characteristics of that produced byhybridoma ATCC CRL 8001, said antibody being in admixture with adiagnostically acceptable carrier.
 14. A method for determining in anindividual the proportion of circulating lymphocytes that are T cellswhich comprises mixing an effective determining amount of a monoclonalantibody with a circulating lymphocyte composition from said individualand determining the proportion of the circulating lymphocytes whichreact with said antibody, and are thus T cells, which antibody:(a)reacts with essentially all normal human peripheral T cells andcutaneous T lymphoma cells, but not with normal human peripheral Bcells, null cells or macrophages; (b) reacts with from about 5% to about10% of normal human thymocytes; (c) reacts with leukemic cells fromhumans with T cell chronic lymphoblastic leukemic but does not reactwith leukemic cells from humans with T cell acute lymphoblasticleukemia, null cell acute lymphoblastic leukemia, or B cell chroniclymphatic leukemia; (d) reacts weakly with the human T cell line HJD-1but does not react with CEM, Laz 191, or HMl; (e) does not react withthe Epstein-Barr virus-transformed human B cell lines Laz 007, Laz 156,Laz 256, or SB; and (f) fixed complement.
 15. A method for determiningin an individual the proportion of circulating lymphocytes that are Tcells which comprises mixing an effective determining amount of antibodyproduced from a hybridoma having the identifying characteristics of OKT3with a circulating lymphocyte composition from said individual anddetermining the proportion of the circulating lymphocytes which reactwith said antibody, and are thus T cells.